1. Technical Field
The present invention is related to obtaining a novel bacterium belonging to the genus Alcaligenes and capable of producing a unique 17.beta.-hydroxysteroid dehydrogenase as well as other steroid-transforming enzymes, to the use of these enzymes for preparative and analytical purposes, and to the description of a novel enzyme cycling assay capable of detecting picomole quantities of compounds with hydroxyl or carbonyl groups. More particularly, the present invention is directed to the isolation of a morphologically distinct bacterium belonging to Alcaligenes genus capable of being induced to produce steroid-transforming enzymes one of which, a 17.beta.-hydroxysteroid dehydrogenase, has unexpected and unique properties. Because of their unique specificity, these enzymes can be used in the preparative transformation of certain substrates to products and for the analytical detection of steroids and other steroid-transforming enzymes.
The invention also describes a general assay for the detection of compounds with hydroxyl or carbonyl groups via the use of dehydrogenases which have specificity for two or more nicotinamide nucleotides or their analogs. A particular assay for the detection of 17.beta.-hydroxyl- and/or 17-ketosteroids employing 17.beta.-hydroxysteroid dehydrogenase from Alcaligenes (supra) is also described.
2. State of the Art
There are several enzymes which can be grouped under the class of hydroxysteroid dehydrogenase (HSD), such as 3.alpha.-, 3.beta.-, 17.beta.-hydroxysteroid dehydrogenases and the like. Certain HSDs from microbial as well as mammalian sources have been isolated. However, enzymes possessing the properties and specificities as described for the 17.beta.-HSD of the present invention have not heretofore been known. Such unique properties of the 17.beta.-HSD of the present invention were neither predictable nor foreseeable from the prior art. Specifically, prior to the present invention, no highly active 17.beta.-HSD was known which is substantially free of 3.beta.-HSD activity, and which catalyzes oxidation of 17.beta.-hydroxyl and reduction of 17-keto groups of both C.sub.19 and C.sub.18 steroids. For instance, the bacterial 17.beta.-HSD of Pseudomonas testosteroni contains significant 3.beta.-HSD activity and even sustained efforts to obtain a 17.beta.-HSD free of ancillary enzyme(s) have not been successful (Talalay, The Enzymes 2nd Ed., Vol. 7, pp. 177-202, Academic Press, NY, 1963; Schultz et al., J. Biol. Chem. 252:3775-3783, 1977). Further, the HSD of Brevibacterium sterolicum described in U.S. Pat. No. 3,776,816 catalyzes both 3.beta.- and 17.beta.HSD activity. In contrast, the 17.beta.-HSD of the present invention, unlike the 3.beta.- and 17.beta.-HSD of either P. testosteroni or B. sterolicum, is substantially free of 3.beta.-HSD activity and will not catalyze the oxidoreduction of 3.beta.-hydroxy- or 3-keto- groups of steroids. Thus, it is a distinctly different enzyme. Similarly, the mammalian placental 17.beta.-HSD (Jarabak et al., J. Biol. Chem. 237:345-357, 1962; Karavolas et al., J. Biol. Chem. 245:4948-4952, 1970) catalyzes the oxido-reduction of phenolic (estrogenic) 17.beta.-hydroxysteroids and does not react with testosterone and other androgenic 17.beta.-hydroxysteroids and 17-ketosteroids to any appreciable degree. In addition, pure placental 17.beta.-HSD was found to have very low specific activity. Thus, finding an enzyme having the selectivity, specificity and high activity as disclosed herein was by no means predictable from the prior art.
The present invention is the first to obtain a 17.beta.-HSD unexpectedly possessing unique properties such as highly selective and stereospecific oxido-reduction of both C.sub.19 (androgenic) and C.sub.18 (estrogenic) steroids at about equal rates. It should also be emphasized that the 17.beta.-HSD of the present invention is inducibly obtained only from the bacteria belonging to the genus Alcaligenes and is not known to be obtainable from any other source. It should be further noted that without such induction, obtaining the enzyme(s) of the present invention in an amount suitable for the utilities disclosed herein is unlikely.
The Alcaligenes sp. also contains distinct, inducible 3.beta.-HSD, and .DELTA..sup.5 -3-ketosteroid isomerase, and may contain other inducible steroid-transforming enzymes. Although 3.beta.-HSDs and ketosteroid isomerases have been described from other sources, it is both convenient and economical to isolate such enzymes from this species of Alcaligenes during the course of the same purification.
The transhydrogenase function of hydroxysteroid dehydrogenases with dual nicotinamide nucleotide specificity has been described whereby catalytic quantities of steroids promote the following reaction (Talalay & Williams-Ashman, Proc. Natl. Acad. Sci. U.S.A., 44, 15, 1958; Talalay, et al., Proc. Natl. Acad. Sci. U.S.A., 44, 862, 1958): EQU NADPH+NAD.sup.30 .revreaction.NADP.sup.30 +NADH
Recently, Nicolas et al. (Clin. Chim. Acta., 92, 1, 1979) have employed the NAD(P)H-linked transhydrogenase function of placental 17.beta.-HSD to detect picogram quantities of 17.beta.-estradiol and estrone in plasma and urine. However, widespread usefulness of this method is limited by the relative difficulty of obtaining highly purified hydroxysteroid dehydrogenases with dual nucleotide specificity from animal sources and because of the low specific activities of such enzymes. Moreover, since placental 17.beta.-HSD is virtually unable to catalyze the oxido-reductions of C.sub.19 steroids, this assay cannot be used to measure testosterone and related steroids. Furthermore, such prior assay is not applicable with the many bacterial hydroxysteroid dehydrogenases which react with NAD(H) but not NADP(H), as is the case with the HSD of the present invention.
The 17.beta.-HSD (from Alcaligenes) of the present invention has specificity for both C.sub.18 (estrogenic) and C.sub.19 (androgenic) steroids. Since this enzyme reacts with NAD but not NADP, a unique assay was developed for these steroids based on the transhydrogenase function of this enzyme which readily promotes hydrogen transfer between NAD and certain NAD-analogs. The transhydrogenase reaction of bacterial hydroxysteroid dehydrogenases with NADH and NAD-analogs was described earlier (P. Talalay, H. G. Williams-Ashman, Recent Progress in Hormone Research, Vol. 16, pages 1-47, 1960) and the assay of the present invention, provides a unique application of the transhydrogenases function to the detection of steroids. This assay is described more fully below and is applicable not only with 17.beta.-HSD of the present invention, but with any hydroxysteroid dehydrogenase of mammalian or bacterial origin which has specificity for NAD and its analogs. Further, the assay of this invention has general application to the detection of non-steroidal compounds with hydroxyl or carbonyl groups via the transhydrogenase function of appropriate dehydrogenases which have dual specificity for NAD and its analogs.
In short, the assay of the present invention provides a simple, inexpensive, one-step procedure, using ordinary colorimetric equipment, for the detection of compounds in biological materials (e.g. serum, urine, tissue extracts, tissue culture fluid). Because of its simplicity, the assay is, of course, adaptable to automation. It is noted that this assay is unique among steroid assays in its ability to simultaneously detect "families" of steroids, and to provide profiles of certain known and unknown steroids in biological fluids.